Inorganic pyrophosphatase (or inorganic diphosphatase, PPase) is an
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...
() that catalyzes the conversion of one ion of
pyrophosphate to two
phosphate
In chemistry, a phosphate is an anion, salt, functional group or ester derived from a phosphoric acid. It most commonly means orthophosphate, a derivative of orthophosphoric acid .
The phosphate or orthophosphate ion is derived from phospho ...
ions.
This is a highly
exergonic
An exergonic process is one which there is a positive flow of energy from the system to the surroundings. This is in contrast with an endergonic process. Constant pressure, constant temperature reactions are exergonic if and only if the Gibbs fr ...
reaction, and therefore can be coupled to unfavorable biochemical transformations in order to drive these transformations to completion.
The functionality of this
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...
plays a critical role in
lipid metabolism (including lipid synthesis and degradation), calcium absorption and bone formation,
and DNA synthesis,
as well as other
biochemical
Biochemistry or biological chemistry is the study of chemical processes within and relating to living organisms. A sub-discipline of both chemistry and biology, biochemistry may be divided into three fields: structural biology, enzymology an ...
transformations.
Two types of
inorganic diphosphatase, very different in terms of both
amino acid sequence and
structure
A structure is an arrangement and organization of interrelated elements in a material object or system, or the object or system so organized. Material structures include man-made objects such as buildings and machines and natural objects such as ...
, have been characterised to date:
soluble and
transmembrane
A transmembrane protein (TP) is a type of integral membrane protein that spans the entirety of the cell membrane. Many transmembrane proteins function as gateways to permit the transport of specific substances across the membrane. They frequentl ...
proton-pumping pyrophosphatases (sPPases and H(
+)-PPases, respectively). sPPases are ubiquitous
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
s that hydrolyse
pyrophosphate to release heat, whereas H
+-PPases, so far unidentified in
animal
Animals are multicellular, eukaryotic organisms in the Kingdom (biology), biological kingdom Animalia. With few exceptions, animals Heterotroph, consume organic material, Cellular respiration#Aerobic respiration, breathe oxygen, are Motilit ...
and
fungal
A fungus ( : fungi or funguses) is any member of the group of eukaryotic organisms that includes microorganisms such as yeasts and molds, as well as the more familiar mushrooms. These organisms are classified as a kingdom, separately from th ...
cells, couple the energy of PPi
hydrolysis
Hydrolysis (; ) is any chemical reaction in which a molecule of water breaks one or more chemical bonds. The term is used broadly for substitution reaction, substitution, elimination reaction, elimination, and solvation reactions in which water ...
to
proton
A proton is a stable subatomic particle, symbol , H+, or 1H+ with a positive electric charge of +1 ''e'' elementary charge. Its mass is slightly less than that of a neutron and 1,836 times the mass of an electron (the proton–electron mass ...
movement across
biological
Biology is the scientific study of life. It is a natural science with a broad scope but has several unifying themes that tie it together as a single, coherent field. For instance, all organisms are made up of cells that process hereditary in ...
membranes.
Structure
Thermostable soluble pyrophosphatase had been isolated from the
extremophile
An extremophile (from Latin ' meaning "extreme" and Greek ' () meaning "love") is an organism that is able to live (or in some cases thrive) in extreme environments, i.e. environments that make survival challenging such as due to extreme temper ...
''
Thermococcus litoralis''. The 3-dimensional structure was determined using
x-ray crystallography
X-ray crystallography is the experimental science determining the atomic and molecular structure of a crystal, in which the crystalline structure causes a beam of incident X-rays to diffract into many specific directions. By measuring the angles ...
, and was found to consist of two
alpha-helices, as well as an
antiparallel closed
beta-sheet. The form of inorganic pyrophosphatase isolated from ''
Thermococcus litoralis'' was found to contain a total of 174
amino acid residue
Protein structure is the molecular geometry, three-dimensional arrangement of atoms in an amino acid-chain molecule. Proteins are polymers specifically polypeptides formed from sequences of amino acids, the monomers of the polymer. A single ami ...
s and have a
hexameric oligomeric
In chemistry and biochemistry, an oligomer () is a molecule that consists of a few repeating units which could be derived, actually or conceptually, from smaller molecules, monomers.Quote: ''Oligomer molecule: A molecule of intermediate relativ ...
organization (Image 1).
Humans possess two genes encoding pyrophosphatase, PPA1 and PPA2.
PPA1 has been assigned to a
gene locus
In genetics, a locus (plural loci) is a specific, fixed position on a chromosome where a particular gene or genetic marker is located. Each chromosome carries many genes, with each gene occupying a different position or locus; in humans, the total ...
on human
chromosome 10,
and PPA2 to
chromosome 4.
Mechanism
Though the precise mechanism of
catalysis
Catalysis () is the process of increasing the rate of a chemical reaction by adding a substance known as a catalyst (). Catalysts are not consumed in the reaction and remain unchanged after it. If the reaction is rapid and the catalyst recyc ...
via inorganic pyrophosphatase in most organisms remains uncertain, site-directed
mutagenesis studies in ''
Escherichia coli
''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escher ...
'' have allowed for analysis of the
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...
active site
In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate (binding site) a ...
and identification of key
amino acid
Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha am ...
s. In particular, this analysis has revealed 17 residues of that may be of functional importance in
catalysis
Catalysis () is the process of increasing the rate of a chemical reaction by adding a substance known as a catalyst (). Catalysts are not consumed in the reaction and remain unchanged after it. If the reaction is rapid and the catalyst recyc ...
.
Further research suggests that the
protonation
In chemistry, protonation (or hydronation) is the adding of a proton (or hydron, or hydrogen cation), (H+) to an atom, molecule, or ion, forming a conjugate acid. (The complementary process, when a proton is removed from a Brønsted–Lowry acid, ...
state of Asp67 is responsible for modulating the reversibility of the
reaction in ''
Escherichia coli
''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escher ...
''. The
carboxylate functional group of this residue has been shown to perform a
nucleophilic attack on the
pyrophosphate substrate
Substrate may refer to:
Physical layers
*Substrate (biology), the natural environment in which an organism lives, or the surface or medium on which an organism grows or is attached
** Substrate (locomotion), the surface over which an organism lo ...
when four
magnesium
Magnesium is a chemical element with the symbol Mg and atomic number 12. It is a shiny gray metal having a low density, low melting point and high chemical reactivity. Like the other alkaline earth metals (group 2 of the periodic ta ...
ion
An ion () is an atom or molecule with a net electrical charge.
The charge of an electron is considered to be negative by convention and this charge is equal and opposite to the charge of a proton, which is considered to be positive by conven ...
s are present. Direct coordination with these four
magnesium
Magnesium is a chemical element with the symbol Mg and atomic number 12. It is a shiny gray metal having a low density, low melting point and high chemical reactivity. Like the other alkaline earth metals (group 2 of the periodic ta ...
ion
An ion () is an atom or molecule with a net electrical charge.
The charge of an electron is considered to be negative by convention and this charge is equal and opposite to the charge of a proton, which is considered to be positive by conven ...
s and
hydrogen bonding interactions with Arg43, Lys29, and Lys142 (all positively charged residues) have been shown to anchor the substrate to the
active site
In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate (binding site) a ...
. The four
magnesium
Magnesium is a chemical element with the symbol Mg and atomic number 12. It is a shiny gray metal having a low density, low melting point and high chemical reactivity. Like the other alkaline earth metals (group 2 of the periodic ta ...
ion
An ion () is an atom or molecule with a net electrical charge.
The charge of an electron is considered to be negative by convention and this charge is equal and opposite to the charge of a proton, which is considered to be positive by conven ...
s are also suggested to be involved in the stabilization of the
trigonal bipyramid
In geometry, the triangular bipyramid (or dipyramid) is a type of hexahedron, being the first in the infinite set of face-transitive bipyramids. It is the dual of the triangular prism with 6 isosceles triangle faces.
As the name suggests, i ...
transition state
In chemistry, the transition state of a chemical reaction is a particular configuration along the reaction coordinate. It is defined as the state corresponding to the highest potential energy along this reaction coordinate. It is often marked wi ...
, which lowers the energetic barrier for the aforementioned
nucleophilic attack.
Several studies have also identified additional
substrates that can act as
allosteric
In biochemistry, allosteric regulation (or allosteric control) is the regulation of an enzyme by binding an effector molecule at a site other than the enzyme's active site.
The site to which the effector binds is termed the ''allosteric site ...
effectors. In particular, the binding of
pyrophosphate (PPi) to the effector site of inorganic pyrophosphatase increases its rate of
hydrolysis
Hydrolysis (; ) is any chemical reaction in which a molecule of water breaks one or more chemical bonds. The term is used broadly for substitution reaction, substitution, elimination reaction, elimination, and solvation reactions in which water ...
at the
active site
In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate (binding site) a ...
.
ATP
ATP may refer to:
Companies and organizations
* Association of Tennis Professionals, men's professional tennis governing body
* American Technical Publishers, employee-owned publishing company
* ', a Danish pension
* Armenia Tree Project, non ...
has also been shown to function as an
allosteric
In biochemistry, allosteric regulation (or allosteric control) is the regulation of an enzyme by binding an effector molecule at a site other than the enzyme's active site.
The site to which the effector binds is termed the ''allosteric site ...
activator in ''
Escherichia coli
''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escher ...
'',
while
fluoride
Fluoride (). According to this source, is a possible pronunciation in British English. is an inorganic, monatomic anion of fluorine, with the chemical formula (also written ), whose salts are typically white or colorless. Fluoride salts typ ...
has been shown to inhibit
hydrolysis
Hydrolysis (; ) is any chemical reaction in which a molecule of water breaks one or more chemical bonds. The term is used broadly for substitution reaction, substitution, elimination reaction, elimination, and solvation reactions in which water ...
of
pyrophosphate in
yeast
Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. The first yeast originated hundreds of millions of years ago, and at least 1,500 species are currently recognized. They are estimated to constitut ...
.
Biological function and significance
The hydrolysis of inorganic
pyrophosphate (PPi) to two
phosphate
In chemistry, a phosphate is an anion, salt, functional group or ester derived from a phosphoric acid. It most commonly means orthophosphate, a derivative of orthophosphoric acid .
The phosphate or orthophosphate ion is derived from phospho ...
ions is utilized in many biochemical pathways to render reactions effectively irreversible.
This process is highly
exergonic
An exergonic process is one which there is a positive flow of energy from the system to the surroundings. This is in contrast with an endergonic process. Constant pressure, constant temperature reactions are exergonic if and only if the Gibbs fr ...
(accounting for approximately a −19kJ change in
free energy), and therefore greatly increases the energetic favorability of reaction system when coupled with a typically less-favorable reaction.
Inorganic pyrophosphatase catalyzes this
hydrolysis
Hydrolysis (; ) is any chemical reaction in which a molecule of water breaks one or more chemical bonds. The term is used broadly for substitution reaction, substitution, elimination reaction, elimination, and solvation reactions in which water ...
reaction in the early steps of
lipid
Lipids are a broad group of naturally-occurring molecules which includes fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, phospholipids, and others. The functions of lipids include ...
degradation, a prominent example of this phenomenon. By promoting the rapid
hydrolysis
Hydrolysis (; ) is any chemical reaction in which a molecule of water breaks one or more chemical bonds. The term is used broadly for substitution reaction, substitution, elimination reaction, elimination, and solvation reactions in which water ...
of
pyrophosphate (PPi), Inorganic pyrophosphatase provides the driving force for the activation of
fatty acids destined for
beta oxidation.
Before
fatty acids can undergo degradation to fulfill the metabolic needs of an organism, they must first be activated via a thioester linkage to
coenzyme A
Coenzyme A (CoA, SHCoA, CoASH) is a coenzyme, notable for its role in the synthesis and oxidation of fatty acids, and the oxidation of pyruvate in the citric acid cycle. All genomes sequenced to date encode enzymes that use coenzyme A as a subs ...
. This process is catalyzed by the enzyme
acyl CoA synthetase, and occurs on the outer
mitochondrial membrane
A mitochondrion (; ) is an organelle found in the cells of most Eukaryotes, such as animals, plants and fungi. Mitochondria have a double membrane structure and use aerobic respiration to generate adenosine triphosphate (ATP), which is used t ...
. This activation is accomplished in two reactive steps: (1) the fatty acid reacts with a molecule of
ATP
ATP may refer to:
Companies and organizations
* Association of Tennis Professionals, men's professional tennis governing body
* American Technical Publishers, employee-owned publishing company
* ', a Danish pension
* Armenia Tree Project, non ...
to form an enzyme-bound
acyl adenylate and
pyrophosphate (PPi), and (2) the sulfhydryl group of CoA attacks the acyl adenylate, forming
acyl CoA and a molecule of
AMP #REDIRECT Amp #REDIRECT Amp
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. Each of these two steps is reversible under biological conditions, save for the additional hydrolysis of PPi by inorganic pyrophosphatase.
provides the driving force for the overall forward activation reaction, and serves as a source of
forms of soluble inorganic pyrophosphatase (sPPase, ) has shown that they differ significantly in both
organization. Despite differing structural components, recent work has suggested a large degree of
data.
-PPase) that appears to be highly conserved; this region primarily consisted of the four early
.